Fluorous Technologies Technical Bulletin February 2005

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New Oligonucleotide purification technology using fluorous affinity tags

Berry & Associates, Inc. recently licensed FTI's patents covering fluorous synthesis and separation.  With fluorous tags and sorbents as their building blocks, they recently developed a line of fluorous-adapted oligonucleotide synthesis and purification products.  Laboratory synthesis of oligonucleotides has been essential to the molecular biology revolution.  Purifcation challenges, however, have historically limited the yield, purity and length of oligonucleotides. 

Phosphoramidites tagged at the 5' terminus with fluorous dimethoxytrityl (FDMT) allow researchers to readily isolate higher yields (70-100%) of oligonucleotides than is possible by other means, especially when long oligonucleotides (even up to 100-mers) are required.  Data presented by Dr. William Pearson at the recent Association of Biolmolecular Resource Facilities conference illustrated high selectivity and recovery in fluorous separation of FDMT-on oligos from failure sequences.  Like fluorous-enhanced peptide synthesis, fluorous oligo synthesis complements conventional solid-phase strategies by applying the affinity tag with the final coupling before cleavage.

Fluorous-tagged phosphoramidited for DNA synthesis and affinity columns are commercially available from Berry & Associates, and F-DMT-Cl is available from both Fluorous Technologies and Berry. 

Recently published research involving fluorous chemistry


  PURIFICATION FOLLOWING MONOALKYLATION OF AMINES  Dr. Kristensen and colleagues at the Danish University of Pharmaceutical Sciences recently reported on the use of fluorous thiol as a deprotecting / scavenging agent following monoalkylation of primary amines [1].  2-nitrobenzenesulfonamides, which are popular as a protecting group in this chemistry, are simulataneously cleaved and fluorous-tagged for easy separation from the product amine by fluorous SPE.  The strategy was further refined to include a solid-supported base; thus all components except the product were immobilized and high purity was achieved.

monoalkyation of amines

SYNTHESIS OF NITROGEN HETEROCYCLES   Dr. Procter and colleagues at the University of Glasgow developed a fluorous-phase Pummerer cyclative-capture strategy for the high-throughput synthesis of N heterocycles [2]. Their work was intended to improve on earlier experiments using a solid-phase strategy, the optimization of which was frustrated by difficulties in monitoring intermediate transformations. Use of a fluorous thiol tag as a solution-phase support not only allowed in-process analysis, but also enabled simple and effective fluorous SPE purification at each step.



FLUOROUS LIBRARY SYNTHESIS   Dr. Zhang and colleagues at Fluorous Technologies recently reported on the synthesis of two libraries of biologically-interesting compounds using fluorous-enhanced strategies. In each case, microwave irradiation was used in combination with fluorous chemistry, improving throughput with both increased reaction speed and reduced separation time. In the first case, multi-component reactions were used in the solution-phase synthesis of a diverse library of bicyclic prolines [3]. A fluorous sulfonyl group played three roles in the synthesis: a) phase tag to facilitate purification, b) phenol protecting group, and c) triflate equivalent to promote cross-coupling.



In the second paper, they reported on the use of parallel synthesis in the production of an N-alkylated dihydropteridinone library beginning with fluorous-tagged amino acids [4]. The fluorous tag was substituted for resin in an earlier-published solid-phase synthesis. In addition to improving the reaction kinetics, adapting the synthesis from resin to fluorous support enabled the use of a more-easily-removed heterogeneous hydrogenation catalyst as well as the use of microwave radiation to promote a cyclization. Intermediates were monitored by LC/MS (not possible on resin), and fluorous SPE purification resulted in excellent purity while avoiding the need for HPLC.



FLUOROUS GRUBBS-HOVEYDA CATALYSTS   Fluorous chemistry originated as a technique for catalysis. Interest in a variety of fluorous-modified catalysts continues to the present, with wide-ranging work on fluorous versions of BINOL, oxazolidinone chiral auxiliaries, palladium and platinum complexes, Wilkinson's catalyst, and numerous others (citations and additional information available on request). In some cases, the value of fluorous separation is in the removal of toxic homogeneous species, while in other cases fluorous techniques are useful in the recovery of high-value materials for re-use.

In an example of the latter, FTI founder Prof. Curran and his co-worker at the University of Pittsburgh developed a light fluorous adaptation of the Grubbs-Hoveyda catalyst, which has rapidly gained recognition for its utility in olefin metathesis [5]. They demonstrated that the fluorous-tagged analog performed essentially the same as the non-fluorous parent, while also featuring simple recovery by fluorous SPE. The recovered fluorous catalyst was able to be re-used repeatedly.
Note to readers with potential interest in fluorous Grubbs-Hoveyda catalysts: A limited license to practice light fluorous chemistry is granted with the sale of products by Fluorous Technologies. However, commercial evaluation / use of this particular application may also be subject to additional patent protections held by others.



CONTINUOUS EXTRACTION OF FLUOROUS-TAGGED SPECIES AFTER CHIRAL RESOLUTION   Dr. Jaenicke and colleagues at the National University of Singapore reported on the use of a membrane contactor to continuously extract fluorous-tagged species following dynamic resolution of secondary alcohols using enzymatic techniques [6]. This topic also recalls earlier work by Prof. Curran, in which fluorous tags were enantioselectively applied within a racemic mixture of 2-napthylethanol, which was then separated using a fluorous triphasic system[7].



Reprints of FTI-authored papers are available on request from FTI

 

Free fluorous chemistry lecture slides

For our colleagues in the academic community, FTI is pleased to offer free PowerPoint lecture slides on fluorous chemistry. The presentation is scientific rather than commercial in nature, and it is intended to be a teaching aid for instruction on fluorous chemistry mechanisms, techniques, and applications. Click here to download.


 

References:

[1] Christensen, C.; Clausen, R. P.; Begtrup, M.; Kristensen, J. l. "Deprotection of 2-nitrobenzenesulfonamides using fluorous and solid-phase reagents" Tetrahedron Letters. 2004. 45, 7991-7993.

[2] McAllister, L.; McCormick, R.; Brand, S.; Procter, D. "A fluorous-phase Pummerer cyclative-capture strategy for the synthesis of nitrogen heterocycles" Angew. Chem. Int. Ed. 2005, 44, 452.

[3] Chen, C.; Zhang, W. "Fluorous synthesis of biaryl-substituted proline analogs by 1,3-dipolar cycloaddition and Suzuki coupling reactions" Tetrahedron Letters. 2005, 46, 1807-1810.

[4] Nagashima, T.; Zhang, W. "Solution-Phase Parallel Synthesis of an N-Alkylated Dihydropteridinone Library from Fluorous Amino Acids " J. Comb. Chem. 2004, 6, 942-949.

[5] Matsugi, M; Curran, D. P. "Synthesis, Reaction, and Recycle of Light Fluorous Grubbs-Hoveyda Catalysts for Alkene Metathesis" J. Org. Chem. 2005. 70(5), 1636-1642.

[6] Teo, E.; , G.; A. Huguet, A.; Jaenicke, S.; Pande, G.; and Zhu, Y. "Process intensification with biocatalysts: Dynamic kinetic resolution and fluorous phase switch with continuous extraction", Catalysis Today. 2004, 97, 4, 263-270.

[7] Luo, Z.; Swaleh, S.; Theil, F.; Curran, D. P. "Resolution of 1-(2-Naphthyl)ethanol by a combination of an enzyme-catalyzed kinetic resolution with a fluorous triphasic separative reaction", Org. Lett. 2002, 4, 2585.